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The next hurdle was the difficulty of consistently culturing the bug
from Crohn's sufferers' intestines.[27]
Although MAP has been independently isolated across three continents --
cultured from Crohn's tissue in California, Texas, France, Australia,
England, the Netherlands, and the Czech republic[27]
-- results are still relatively sparse and many labs have reported not
being able to culture it at all.[115]
This is not surprising.[24]

In order to isolate a specific bug from the multitude that exist
naturally in the intestine, one has to devise a decontamination technique
that kills other bacteria without harming the target bacterium, in this
case MAP. Without their protective cell walls, however, cell wall
deficient forms are almost impossible to culture because of the caustic
processing techniques required to isolate them.[22]

Even once isolated, MAP is very difficult to grow.[68]
Researchers have been trying since 1952 to grow mycobacteria from
surgically removed Crohn's disease tissue.[46]
It is thought that Chiodini succeeded where others had failed because of
his many years of experience, combined with access to modern culture
techniques and years of patient work.[126]
Some human isolates took up to six years to grow, even under extremely
precise culture and decontamination conditions.66 Earlier researchers
failed to meet these stringent standards for culturing the bacteria.[83]

Even modern labs have been found to be relying on faulty study design.[66]
Moreover, the differences in methods used between labs can be vast.[24]
Some labs still use fixed or frozen specimens or use only surface tissues
from superficial biopsies, when it's been shown that one should optimally
use fresh[66] resected
tissue, as MAP tends to be found deep in the intestinal wall.[166]
Some labs working with nonspheroplast forms of MAP from cattle haven't
even been able to grow it. Even under the best circumstances, MAP is a
tough bug to grow.[67]

To this day, many infectious agents have eluded our attempts to grow
them in labs at all. For example, scientists have never been able to
isolate Mycobacterium leprae, the microbe responsible for leprosy. Even
Campylobacter, which we now know as the most significant bacteria in food
poisoning, wasn't identified as a human pathogen until the 1970s, when
culturing techniques enabling isolation were finally developed.[101]

Complicating attempts to culture the bug in Crohn's, there seem to be
very few MAP actually involved in the disease process. This has a parallel
in other animals -- MAP bacteria in sheep and goat paratuberculosis are
often sparse or even undetectable[147]
-- and in other mycobacterial human diseases like a type of leprosy in
which just a few mycobacteria are capable of triggering a pathological
immune response.[67]

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